Method for biotinylation of Jo-1 antigen and anti-Jo-1 antibody detection kitTechnical Field
The invention belongs to the field of antibody detection, and particularly relates to a method for biotinylating a Jo-1 antigen and an anti-Jo-1 antibody detection kit.
Background
anti-Jo-1 antibodies are commonly found in Polymyositis (PM), and the positive detection rate can reach 40% -50%; in patients with PM overlapping DM (dermatomyositis), the positive detection rate of anti-Jo-1 antibody is 25%; in DM alone, the detection rate of anti-Jo-1 antibodies was less than 10%; in other autoimmune diseases, the anti-Jo-1 antibody is negative, and thus the anti-Jo-1 antibody is specific for diagnosing PM. In addition, in patients with PM overlapping scleroderma, the positive rate detection rate of anti-Jo-1 antibodies can be as high as 85%; the detection rate of the positive rate of the anti-Jo-1 antibody in patients with Progressive Systemic Sclerosis (PSS) overlapped with PM is about 25%, and the positive rate of the anti-Jo-1 antibody in patients with PM accompanied by interstitial fibrosis of the lung can reach 60%.
At present, detection methods of anti-Jo-1 antibodies are reported, and the detection methods of the diseases mainly comprise an indirect immunofluorescence method and an enzyme-linked immunosorbent assay method, but the methods have the defects. The indirect immunofluorescence method can not distinguish nonspecific reaction according to the molecular weight when analyzing the result; the operation is relatively complex, a fluorescence microscope with high price is required, the popularization is difficult in many primary hospitals, and the fluorescence microscope is not suitable for laboratories and diagnosis hospitals with large sample sizes; the background in the fluorescent measurement is high, and the fluorescent immunization technique has a certain difficulty in quantitative measurement; the result determination requires experienced professionals, and the objectivity of the analysis result is insufficient. The detection range and the sensitivity of the ELISA method are low; the detection time is long, more than 2 hours are generally required for completing one test, and the clinical rapid diagnosis requirement cannot be completely met; the random sample injection detection cannot be carried out, and hysteresis exists in the detection result; most of the detection of the instrument can only qualitatively judge the detected antibody in the sample, and the detection result can not monitor the disease.
In order to solve the above problems, in the previous studies, we developed a magnetic particle chemiluminescence detection kit (CN 109085343A) for determining anti-Jo-1 antibody, which has the advantages of good stability, high sensitivity, good repeatability and the like compared with the previous kit by adding a certain amount of tRNA and RNase inhibitor to antigen and increasing the detection sensitivity of Jo-1 antibody through the tRNA and the complex formed by combining tRNA with Jo-1 antigen. However, tRNA and RNase inhibitors are costly and require high storage conditions, and the anti-protozoal processes in the CN109085343A and other current kits are to react activated biotin with amino groups on antigen proteins and link the biotin and the proteins together, so that along with the research, in the process of the Jo-1 antigen biotinylation, the efficiency is low and the ideal level cannot be reached, so that the clinical compliance of the kit is not ideal. Therefore, there is a need to develop a method that can increase the biotinylation of Jo-1 antigen, and also increase the clinical compliance of the kit without the use of tRNA and RNase inhibitors.
Disclosure of Invention
The invention aims to provide a high-efficiency Jo-1 antigen biotinylation method.
Another object of the present invention is to provide an anti-Jo-1 antibody detection kit that can have a high clinical compliance without using tRNA and RNase inhibitors.
In order to solve the technical problems, the invention adopts the following technical scheme:
a method for biotinylating Jo-1 antigen, which converts the sulfhydryl group of Jo-1 antigen into amino group to obtain treated Jo-1 antigen, and then couples the treated Jo-1 antigen with biotin to obtain biotin-labeled Jo-1 antigen.
The inventor adds a step of Jo-1 antigen treatment in a Jo-1 antigen biotinylation method for the first time, converts sulfhydryl groups of the Jo-1 antigen into amino groups, so as to increase the number of the amino groups, improve the probability of the contact reaction of the Jo-1 antigen and biotin, and further improve the biotinylation efficiency of the Jo-1 antigen.
In the invention, 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid sulfosuccinimidyl ester sodium salt is called sulfo SMCC for short; 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester is abbreviated as SMCC.
Preferably, the Jo-1 antigen is reacted with sulfoSMCC and lysine to provide the treated Jo-1 antigen.
Further preferably, said sulfosmcc and said lysine are reacted to provide an amino-labeled SMCC, and said amino-labeled SMCC is then reacted with Jo-1 antigen to provide said treated Jo-1 antigen.
The Jo-1 antigen processing method has the advantages of easily available raw materials, simple processing method and mild conditions.
Still further preferably, the sulfosmcc is dosed in the form of a 0.5 to 2mol/L solution of sulfosmcc, the solvent of which is one or more of dimethyl sulfoxide, N-Dimethylformamide (DMF), or a phosphate buffer (PBS buffer) having a pH of 7 to 7.5.
Still more preferably, the concentration of the sulfoSMCC solution is 0.8 to 1.5mol/L.
Still more preferably, the lysine is fed in the form of a lysine solution having a concentration of 0.5 to 2mol/L, and the solvent of the lysine solution is a phosphate buffer having a pH of 7 to 7.5.
Still more preferably, the concentration of the lysine solution is 0.8 to 1.5mol/L.
Still further preferably, the feeding mole ratio of the sulfo SMCC to the lysine is 1:5-15.
Still more preferably, the feeding mole ratio of the sulfo SMCC to the lysine is 1:8-12.
Still more preferably, the Jo-1 antigen is administered as a solution of Jo-1 antigen, and the solution of Jo-1 antigen is in a phosphate buffer having a pH of 7 to 7.5.
Still further preferably, the feed ratio of said amino-labeled SMCC to said Jo-1 antigen is 1/(30-80) mol/g.
Still more preferably, the feed ratio of said amino-labeled SMCC to said Jo-1 antigen is 1/(40-70) mol/g.
Still more preferably, the reaction temperature of the sulfosmcc and the lysine is 20 to 30 ℃.
The reaction of the sulfo SMCC and the lysine is carried out at room temperature.
Still more preferably, the reaction time of the sulfosmcc and the lysine is 1 to 5 hours.
Still more preferably, the reaction time of the sulfosmcc and the lysine is 1 to 3 hours.
Still further preferably, the reaction temperature of the reaction of the amino-labeled SMCC and the Jo-1 antigen is between 0 and 10 ℃.
Still more preferably, the reaction temperature of the reaction of the amino-labeled SMCC and the Jo-1 antigen is between 2 and 8 ℃.
Still further preferably, the reaction time of the amino-labeled SMCC and the Jo-1 antigen is 20 to 30 hours.
Still more preferably, the reaction time of the amino-labeled SMCC and the Jo-1 antigen is 20 to 24 hours.
Further preferably, the method for preparing the treated Jo-1 antigen comprises the following specific steps:
step 1: mixing 0.5-2 mol/L sulfo SMCC solution and 0.5-2 mol/L lysine solution, and reacting at 20-30 ℃ for 1-5 h;
step 2: adding Jo-1 antigen solution with the mass concentration of 0.15-0.5 mg/mL, and reacting for 20-30 h at the temperature of 0-10 ℃;
step 3: dialyzing the reacted reaction solution to remove unreacted sulfo SMCC and unreacted amino-labeled SMCC to obtain a treated Jo-1 antigen;
wherein, the solvent of the sulfo SMCC solution is dimethyl sulfoxide; the solvent of the lysine solution and the Jo-1 antigen solution is phosphate buffer solution with the pH value of 7-7.5; the volume ratio of the sulfo SMCC solution to the lysine solution to the Jo-1 antigen solution is 1:5-15:60-120.
Still more preferably, the volume ratio of the sulfoSMCC solution, the lysine solution and the Jo-1 antigen solution is 1:8-12:80-110.
Still more preferably, the phosphate buffer of the present invention includes 0.08 to 0.2M sodium phosphate and 0.01 to 0.2M NaCl.
Preferably, the biotin is biotin activated with N-hydroxysuccinimide.
An anti-Jo-1 antibody assay kit comprising a first reagent that is a biotin-labeled Jo-1 antigen prepared by the method of biotinylating the Jo-1 antigen.
Preferably, the kit further comprises a second reagent, a magnetic particle reagent, a chemiluminescent substrate, a calibrator, a quality control product and a cleaning solution, wherein the second reagent comprises an alkaline phosphatase-labeled anti-human IgG antibody.
Further preferably, the particle diameter of the magnetic particles is 0.1-0.5 μm, the surface of the magnetic particles contains streptavidin groups, and the chemiluminescent substrate is adamantane.
In the present invention, the methods for preparing the second reagent, the calibrator and the quality control material and the method for detecting the kit refer to patent CN109085343a.
Compared with the prior art, the invention has the following advantages:
the invention provides a process for converting sulfhydryl of a Jo-1 antigen into amino, so that the number of amino on the surface of the Jo-1 antigen is increased, the probability of reaction of the Jo-1 antigen and biotin is improved when the Jo-1 antigen is biotinylated, the biotinylation efficiency is increased, the raw materials are easy to obtain, the method is simple, and the cost is low.
Drawings
FIG. 1 shows the principle of chemiluminescent detection.
FIG. 2 is a linear regression curve of the kit of example 1.
FIG. 3 is a schematic diagram showing the stability results of the kit of example 1.
Detailed Description
The invention is further described below with reference to examples. The present invention is not limited to the following examples. The implementation conditions adopted in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions which are not noted are conventional conditions in the industry. The technical features of the various embodiments of the present invention may be combined with each other as long as they do not collide with each other.
In the existing Jo-1 antigen biotinylation process, the Jo-1 antigen is directly biotinylated, the efficiency is low, and the clinical compliance of the kit is not ideal, so that the efficiency of Jo-1 antigen biotinylation needs to be improved.
With the intensive research we analyzed that the reason why the biotinylation efficiency of Jo-1 antigen was low might be that the number of exposed amino groups was insufficient, and that Jo-1 antigen could not react sufficiently with activated biotin. Therefore, we have devised a method of biotinylation of Jo-1 antigen, converting sulfhydryl groups on the surface of Jo-1 antigen into amino groups to obtain treated Jo-1 antigen, increasing the number of amino groups of Jo-1 antigen, increasing the efficiency of the binding reaction of Jo-1 antigen with biotin, and then coupling the treated Jo-1 antigen with biotin to obtain biotin-labeled Jo-1 antigen.
Specifically, jo-1 antigen is reacted with sulfoSMCC and lysine to give the treated Jo-1 antigen.
Further, the sulfoSMCC and the lysine are reacted to provide an amino-labeled SMCC, and the amino-labeled SMCC is then reacted with a Jo-1 antigen to provide the treated Jo-1 antigen.
According to some embodiments, the specific steps of biotinylation of the Jo-1 antigen are:
step 1: mixing 0.5-2 mol/L sulfo SMCC solution and 0.5-2 mol/L lysine solution, and reacting at 20-30 ℃ for 1-5 h;
step 2: adding Jo-1 antigen solution with the mass concentration of 0.15-0.5 mg/mL, and reacting for 20-30 h at the temperature of 0-10 ℃;
step 3: dialyzing the reacted reaction solution to remove unreacted sulfo SMCC and unreacted amino-labeled SMCC to obtain a treated Jo-1 antigen;
wherein, the solvent of the sulfo SMCC solution is dimethyl sulfoxide; the solvent of the lysine solution and the Jo-1 antigen solution is phosphate buffer solution with the pH value of 7-7.5; the volume ratio of the sulfo SMCC solution to the lysine solution to the Jo-1 antigen solution is 1:5-15:60-120.
In the present invention, biotin is biotin activated with N-hydroxysuccinimide.
According to some embodiments, an anti-Jo-1 antibody detection kit comprises a first reagent, a second reagent, a magnetic particle reagent, a chemiluminescent substrate, a calibrator, a quality control, and a wash solution, wherein the first reagent is a biotin-labeled Jo-1 antigen prepared by the method of biotinylation of the Jo-1 antigen; the second reagent comprises an alkaline phosphatase-labeled anti-human IgG antibody; the particle size of the magnetic particles is 0.1-0.5 mu m, the surfaces of the magnetic particles contain streptavidin groups, and the chemiluminescent substrate is adamantane.
The invention can adopt a full-automatic nano magnetic particle chemiluminescence detection platform, and belongs to quantitative detection. The Alkaline Phosphatase (AP) -Adamantane (AMPPD) system has the advantages of good stability, high sensitivity, good repeatability and the like. Meanwhile, the total time required for completing one test is approximately within 50 minutes, so that the detection time is greatly shortened, the operation is simple and convenient, and the full automation of detection is truly realized.
The kit of the invention can help to evaluate polymyositis, and is particularly used for differential diagnosis of polymyositis in vitro. Diagnosis is realized by analyzing the biochemical marker, the concentration of Jo-1 resistance in a sample is measured, and the measured concentration is related to diagnosis of polymyositis, so that the severity of the disease is accurately diagnosed, doctors are guided to take medicines, and the early diagnosis and treatment of patients are guaranteed.
In the examples below, the conditions under which they are not specified are those conventional in the industry, and reagents are commercially available. Sulfo SMCC was purchased from Thermo.
Example 1
The main components of the anti-Jo-1 antibody detection kit are as follows:
table 1: main component of anti-Jo-1 antibody detection kit
The Jo-1 antigen biotinylation method is as follows:
1) 1mmol of lysine (146.19 mg) was dissolved in 1ml of 0.1M sodium phosphate, 0.15M NaCl, pH 7.2 phosphate buffer to give a 1mol/L lysine solution.
2) 1mmol of sulfoSMCC (436.37 mg), 1mol/L of sulfoSMCC solution was dissolved in 1ml of DMSO.
3) To 1ml of 1mol/L lysine solution was added 0.1ml of 1mol/L sulfoSMCC solution, and immediately after the addition, the mixture was homogenized. And uniformly mixing at room temperature for reaction for 2 hours to obtain the SMCC solution after amino labeling.
4) 3mg of Jo-1 antigen was dissolved in 9ml of 0.1M sodium phosphate, 0.15M NaCl, pH 7.2 phosphate buffer. 5) To 9ml of the Jo-1 antigen solution, 1ml of the above-mentioned amino-labeled SMCC solution was added, and the mixture was reacted at 2 to 8℃for 24 hours.
6) The reacted Jo-1 antigen was finally obtained by dialysis against unreacted SMCC and removal of the unreacted SMCC.
7) Mixing Jo-1 antigen with biotin activated by N-hydroxysuccinimide, and uniformly mixing at 20-30 ℃ for reaction for 20-40 min;
8) Adding a tris buffer solution with the mass concentration of 0.04-0.06 mol/L, uniformly mixing and reacting for 20-40 min at the temperature of 25-35 ℃, and then adding glycerol to obtain the biotinylated Jo-1 antigen;
9) The biotinylated Jo-1 antigen was diluted to a mixed solution with a concentration of 0.5 to 1ug/mL with a phosphate buffer solution having a pH of 7 to 8 and a mass concentration of 0.01 to 0.02 mol/L.
Comparative example 1
Substantially the same as in example 1, except that: the first reagent was prepared without the Jo-1 antigen sulfhydryl transamination step.
The full chemistry luminescence tester test procedure disclosed in patent CN109085343a was used for testing, the test results were as follows:
sample alignment of example 1 and comparative example 1
Negative and positive coincidence rate: the amounts of 328 cases of clinical serum anti-Jo-1 antibodies were detected using the kits of example 1 and comparative example 1, respectively, and were compared clinically with similar products of foreign well-known companies. Table 2 shows that the negative compliance of the anti-Jo-1 antibody kit after conversion of the thiol group of the antigen to an amino group was 99.56% (226/227), the positive compliance was 100.00% (101/101), and Table 3 shows that the negative compliance of the anti-Jo-1 antibody kit after conversion of the thiol group of the antigen to an amino group was 99.56% (226/227), the positive compliance was 90.00% (91/101). The clinical compliance rate of the kit of example 1 was significantly improved over comparative example 1.
Table 2: anti-Jo-1IgG clinical sample alignment of example 1
Table 3: anti-Jo-1IgG clinical sample alignment of comparative example 1
Other Performance test of the kit of example 1
(1) Sensitivity: the LOD of the anti-Jo-1 antibody detection kit is 0.013RU/mL, and the sensitivity of the ELISA method is 2RU/mL.
(2) Linearity: a dilution of 1/2, 1/4, 1/8, 1/16, 1/40, 1/80, 1/200, 1/400 of the high-value serum was tested using the anti-Jo-1 antibody kit of example 1, and the theoretical concentration was linearly regressed with the actual concentration tested, as shown in FIG. 2.
(3) Accuracy: the accuracy was assessed by sample recovery. One high serum, one median serum and one low serum were used according to 1:9 was added to two base sera and their concentrations calculated. The results are shown in Table 4, and the recovery rate of the added sample of the blood serum is 98% -105%.
Additive recovery experiments (sample recovery = post-additive template value/(0.1 sample a+0.9 sample B) 100%
TABLE 4 Table 4
(4) Precision: the quality control products with two different concentrations are detected twice a day, in the morning and afternoon, 4 times of detection are carried out each time, the total detection is carried out for 5 days, each concentration is detected for 40 times, the variation coefficient is calculated, the result is shown in Table 5, and the variation coefficient is shown to be within 5%.
TABLE 5
(5) Stability: after the anti-Jo-1 antibody detection kit of the present invention was left at 37℃for 1 day, 4 days and 7 days, the signal retention was measured, respectively, and the results are shown in Table 6 and FIG. 3. The results showed that the signal retention was 95.2% after 1 day at 37℃and 92.8% after 4 days and 89.4% after 7 days. The anti-Jo-1 antibody detection kit is stable and meets clinical requirements.
TABLE 6
(6) Specificity: the serum with different concentration values of high, medium and low is added with bilirubin (table 7), hemoglobin (table 8) and triglyceride (table 9) for detection, and the result shows that the additive substance has no influence on the detection result of the anti-Jo-1 antibody detection kit.
TABLE 7
TABLE 8
TABLE 9
The present invention has been described in detail with the purpose of enabling those skilled in the art to understand the contents of the present invention and to implement the same, but not to limit the scope of the present invention, and all equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of the present invention.